Protease-activated receptors (PARs) are G protein-coupled receptors that are activated by proteolytical cleavage of the amino-terminus and thereby act as sensors for extracellular proteases. While coagulation proteases activate PARs to regulate hemostasis, thrombosis, and cardiovascular function, PAR2 is also activated in extravascular locations by a broad array of serine proteases, including trypsin, tissue kallikreins, coagulation factors VIIa and Xa, mast cell tryptase, and transmembrane serine proteases. Administration of PAR2-specific agonistic and antagonistic peptides, as well as studies in PAR2 knockout mice, identified critical functions of PAR2 in development, inflammation, immunity, and angiogenesis. Here, we review these roles of PAR2 with an emphasis on the role of coagulation and other extracellular protease pathways that cleave PAR2 in epithelial, immune, and neuronal cells to regulate physiological and pathophysiological processes.

The tissue factor (TF) pathway plays a central role in hemostasis and thrombo-inflammatory diseases. Although structure-function relationships of the TF initiation complex are elucidated, new facets of the dynamic regulation of TF's activities in cells continue to emerge. Cellular pathways that render TF non-coagulant participate in signaling of distinct TF complexes with associated proteases through the protease-activated receptor (PAR) family of G protein-coupled receptors. Additional co-receptors, including the endothelial protein C receptor (EPCR) and integrins, confer signaling specificity by directing subcellular localization and trafficking. We here review how TF is switched between its role in coagulation and cell signaling through thiol-disulfide exchange reactions in the context of physiologically relevant lipid microdomains. Inflammatory mediators, including reactive oxygen species, activators of the inflammasome, and the complement cascade play pivotal roles in TF procoagulant activation on monocytes, macrophages and endothelial cells. We furthermore discuss how TF, intracellular ligands, co-receptors and associated proteases are integrated in PAR-dependent cell signaling pathways controlling innate immunity, cancer and metabolic inflammation. Knowledge of the precise interactions of TF in coagulation and cell signaling is important for understanding effects of new anticoagulants beyond thrombosis and identification of new applications of these drugs for potential additional therapeutic benefits.

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The tissue factor (TF) pathway serves both hemostasis and cell signaling, but how cells control these divergent functions of TF remains incompletely understood. TF is the receptor and scaffold of coagulation proteases cleaving protease-activated receptor 2 (PAR2) that plays pivotal roles in angiogenesis and tumor development. Here we demonstrate that coagulation factor VIIa (FVIIa) elicits TF cytoplasmic domain-dependent proangiogenic cell signaling independent of the alternative PAR2 activator matriptase. We identify a Lys-Gly-Glu (KGE) integrin-binding motif in the FVIIa protease domain that is required for association of the TF-FVIIa complex with the active conformer of integrin β1. A point mutation in this motif markedly reduces TF-FVIIa association with integrins, attenuates integrin translocation into early endosomes, and reduces delayed mitogen-activated protein kinase phosphorylation required for the induction of proangiogenic cytokines. Pharmacologic or genetic blockade of the small GTPase ADP-ribosylation factor 6 (arf6) that regulates integrin trafficking increases availability of TF-FVIIa with procoagulant activity on the cell surface, while inhibiting TF-FVIIa signaling that leads to proangiogenic cytokine expression and tumor cell migration. These experiments delineate the structural basis for the crosstalk of the TF-FVIIa complex with integrin trafficking and suggest a crucial role for endosomal PAR2 signaling in pathways of tissue repair and tumor biology.

Objective Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine (PS) and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a non-coagulant state and to generating prothrombotic TF remains incompletely understood. Approach and Results Here we study activation of TF in primary macrophages by a combination of pharmacological, genetic and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. Following cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles (MV). TF cell surface availability for release onto MV is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, MV proteome analysis identifies activation of Gαi2 as a participating factor in the release of MV with prothrombotic activity in flowing blood. ATP not only prevents TF and PS internalization, but furthermore induces TF conversion to a conformation with high affinity for its ligand, FVIIa. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant PS, the resulting TF+ MV distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for MV generated by thrombo-inflammatory P2rx7 activation. Conclusions These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic MV from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional MV-based diagnostic approaches.